Mesenchymal Stem Cell-Secreted Exosomes and Soluble Signals Regulate Breast Cancer Metastatic Dormancy: Current Progress and Future Outlook.

Breast cancer is most common in women, and in most cases there is no evidence of spread and the primary tumor is removed, resulting in a 'cure'. However, in 10% to 30% of these women, distant metastases recur after years to decades. This is due to breast cancer cells disseminating to distant organs and lying quiescent. This is called metastatic dormancy. Dormant cells are generally resistant to chemotherapy, hormone therapy and immunotherapy as they are non-cycling and receive survival signals from their microenvironment. In this state, they are clinically irrelevant. However, risk factors, including aging and inflammation can awaken dormant cells and cause breast cancer recurrences, which may happen even more than ten years after the primary tumor removal. How these breast cancer cells remain in dormancy is being unraveled. A key element appears to be the mesenchymal stem cells in the bone marrow that have been shown to promote breast cancer metastatic dormancy in recent studies. Indirect co-culture, direct co-culture and exosome extraction were conducted to investigate the modes of signal operation. Multiple signaling molecules act in this process including both protein factors and microRNAs. We integrate these studies to summarize current findings and gaps in the field and suggest future research directions for this field.

Modeling Tumor Cell Dormancy in an Ex Vivo Liver Metastatic Niche.

Despite decades of research into metastatic disease, our knowledge of the mechanisms governing dormancy are still limited. Unraveling the process will aid in developing effective therapies to either maintain or eliminate these dormant cells and thus prevent them from emerging into overt metastatic disease. To study the behavior of dormant tumor cells-mechanisms that promote, maintain, and disrupt this state-we utilize the Legacy LiverChip®, an all-human ex vivo hepatic microphysiological system. This complex, bioengineered system is able to recreate metastatic disease that is reflective of the human situation and is among only a handful of systems able to mimic spontaneous tumor cell dormancy. The dormant subpopulation reflects the defining traits of cellular dormancy-survival in a foreign microenvironment, chemoresistance, and reversible growth arrest. This microphysiological system has and continues to provide critical insights into the biology of dormant tumor cells. It also serves as an accessible tool to identify new therapeutic strategies targeting dormancy and concurrently evaluate the efficacy of therapeutic agents as well as their metabolism and dose-limiting toxicity.

Interferon-γ increases sensitivity to chemotherapy and provides immunotherapy targets in models of metastatic castration-resistant prostate cancer.

Interferon-γ (IFNγ) is a cytokine with limited evidence of benefit in cancer clinical trials to date. However, it could potentially play a role in potentiating anti-tumor immunity in the immunologically "cold" metastatic castration-resistant prostate cancer (mCRPC) by inducing antigen presentation pathways and concurrently providing targets for immune checkpoint blockade therapy. Moreover, it could additionally increase sensitivity to chemotherapy based on its pleiotropic effects on cell phenotype. Here, we show that IFNγ treatment induced expression of major histocompatibility class-I (MHC-I) genes and PD-L1 in prostate cancer cells in vitro. Furthermore, IFNγ treatment led to a decrease in E-cadherin expression with a consequent increase in sensitivity to chemotherapy in vitro. In an in vivo murine tumor model of spontaneous metastatic prostate cancer, IFNγ systemic pretreatment upregulated the expression of HLA-A and decreased E-cadherin expression in the primary tumor, and more importantly in the metastatic site led to increased apoptosis and limited micrometastases in combination with paclitaxel treatment compared to diffuse metastatic disease in control and monotherapy treatment groups. These findings suggest that IFNγ may be useful in combinatorial regimens to induce sensitivity to immunotherapy and chemotherapy in hepatic metastases of mCRPC.

Atorvastatin facilitates chemotherapy effects in metastatic triple-negative breast cancer.

BACKGROUND: Metastatic triple-negative breast cancer (mTNBC) is treated mainly with chemotherapy. However, resistance frequently occurs as tumours enter dormancy. Statins have been suggested as effective against cancer but as they prolong and promote dormancy, it is an open question of whether the concomitant use would interfere with chemotherapy in primary and mTNBC. We examined this question in animal models and clinical correlations. METHODS: We used a xenograft model of spontaneous metastasis to the liver from an ectopic tumour employing a mTNBC cell line. Atorvastatin was provided to sensitise metastatic cells, followed by chemotherapy. The effects of statin usage on outcomes in women with metastatic breast cancer was assessed respectively by querying a database of those diagnosed from 1999 to 2019. RESULTS: Atorvastatin had limited influence on tumour growth or chemotherapy effects in ectopic primary tumours. Interestingly, atorvastatin was additive with doxorubicin (but not paclitaxel) when targeting liver metastases. E-cadherin-expressing, dormant, breast cancer cells were resistant to the use of either statins or chemotherapy as compared to wild-type cells; however, the combination of both did lead to increased cell death. Although prospective randomised studies are needed for validation, our retrospective clinical analysis suggested that patients on statin treatment could experience prolonged dormancy and overall survival; still once the tumour recurred progression was not affected by statin use. CONCLUSION: Atorvastatin could be used during adjuvant chemotherapy and also in conjunction with metastatic chemotherapy to reduce mTNBC cancer progression. These preclinical data establish a rationale for the development of randomised studies.

IP-10 (CXCL10) Can Trigger Emergence of Dormant Breast Cancer Cells in a Metastatic Liver Microenvironment.

Metastatic breast cancer remains a largely incurable and fatal disease with liver involvement bearing the worst prognosis. The danger is compounded by a subset of disseminated tumor cells that may lie dormant for years to decades before re-emerging as clinically detectable metastases. Pathophysiological signals can drive these tumor cells to emerge. Prior studies indicated CXCR3 ligands as being the predominant signals synergistically and significantly unregulated during inflammation in the gut-liver axis. Of the CXCR3 ligands, IP-10 (CXCL10) was the most abundant, correlated significantly with shortened survival of human breast cancer patients with metastatic disease and was highest in those with triple negative (TNBC) disease. Using a complex ex vivo all-human liver microphysiological (MPS) model of dormant-emergent metastatic progression, CXCR3 ligands were found to be elevated in actively growing populations of metastatic TNBC breast cancer cells whereas they remained similar to the tumor-free hepatic niche in those with dormant breast cancer cells. Subsequent stimulation of dormant breast cancer cells in the ex vivo metastatic liver MPS model with IP-10 triggered their emergence in a dose-dependent manner. Emergence was indicated to occur indirectly possibly via activation of the resident liver cells in the surrounding metastatic microenvironment, as stimulation of breast cancer cells with exogenous IP-10 did not significantly change their migratory, invasive or proliferative behavior. The findings reveal that IP-10 is capable of triggering the emergence of dormant breast cancer cells within the liver metastatic niche and identifies the IP-10/CXCR3 as a candidate targetable pathway for rational approaches aimed at maintaining dormancy.

Tea tree oil extract causes mitochondrial superoxide production and apoptosis as an anticancer agent, promoting tumor infiltrating neutrophils cytotoxic for breast cancer to induce tumor regression.

The antitumor activity of the tea tree oil (TTO) derived product, Melaleuca Alternifolia Concentrate (MAC) was characterized mechanistically at the molecular and cellular level. MAC was analyzed for its anticancer activity against human prostate (LNCaP) and breast (MCF-7) cancer cell lines growing in vitro. MAC (0.02-0.06% v/v) dose-dependently induced the intrinsic (mitochondrial) apoptotic pathway in both the LNCaP and MCF-7 cell lines, involving increased mitochondrial superoxide production, loss of mitochondrial membrane potential (MMP), caspase 3/7 activation, as well as the presence of TUNEL+ and cleaved-PARP+ cell populations. At concentrations of 0.01-0.04% v/v, MAC caused cell cycle arrest in the G0/1-phase, as well as autophagy. The in vivo anticancer actions of MAC were examined as a treatment in the FVB/N c-Neu murine model for spontaneously arising breast cancers. Intratumoral MAC injections (1-4% v/v) significantly suppressed tumor progression in a dose-dependent manner and was associated with greater levels of tumor infiltrating neutrophils exhibiting anticancer cytotoxic activity. Induction of breast cancer cell death by MAC via the mitochondrial apoptotic pathway was also replicated occurring in tumors treated in vivo. In conclusion, our data highlights the potential for the Melaleuca-derived MAC product inducing anticancer neutrophil influx, supporting its application as a novel therapeutic agent.

Integrative microphysiological tissue systems of cancer metastasis to the liver.

The liver is the most commonly involved organ in metastases from a wide variety of solid tumors. The use of biologically and cellularly complex liver tissue systems have shown that tumor cell behavior and therapeutic responses are modulated within the liver microenvironment and in ways distinct from the behaviors in the primary locations. These microphysiological systems have provided unexpected and powerful insights into the tumor cell biology of metastasis. However, neither the tumor nor the liver exist in an isolated tissue situation, having to function within a complete body and respond to systemic events as well as those in other organs. To examine the influence of one organ on the function of other tissues, microphysiological systems are being linked. Herein, we discuss extending this concept to tumor metastases by integrating complex models of the primary tumor with the liver metastatic environment. In addition, inflammatory organs and the immune system can be incorporated into these multi-organ systems to probe the effects on tumor behavior and cancer treatments.

Modeling the Complexity of the Metastatic Niche Ex Vivo.

Cancer mortality predominantly results from distant metastases that are undetectable at diagnosis and escape initial therapies to lie as dormant micrometastases for years. To study the behavior of micrometastases-how they resist initial treatments and then awaken from a dormant state-we utilize the Legacy LiverChip®, an all-human ex vivo hepatic microphysiological system. The functional liver bioreactor, comprising hepatocytes and non-parenchymal cells in a 3D microperfused culture format, mimics the dormant-emergent metastatic progression observed in human patients: (a) a subpopulation of cancer cells spontaneously enter dormancy, (b) cycling cells are eliminated by standard chemotherapies, while quiescent dormant cells remain, and (c) chemoresistant dormant cells can be stimulated to emerge. The system effluent and tissue can be queried for proteomic and genomic data, immunofluorescent imaging as well as drug efficacy and metabolism. This microphysiological system continues to provide critical insights into the biology of dormant and re-emergent micrometastases and serves as an accessible tool to identify new therapeutic strategies targeting the various stages of metastasis, while concurrently evaluating antineoplastic agent efficacy for metastasis, metabolism, and dose-limiting toxicity.

A Perspective on Therapeutic Pan-Resistance in Metastatic Cancer.

Metastatic spread represents the leading cause of disease-related mortality among cancer patients. Many cancer patients suffer from metastatic relapse years or even decades after radical surgery for the primary tumor. This clinical phenomenon is explained by the early dissemination of cancer cells followed by a long period of dormancy. Although dormancy could be viewed as a window of opportunity for therapeutic interventions, dormant disseminated cancer cells and micrometastases, as well as emergent outgrowing macrometastases, exhibit a generalized, innate resistance to chemotherapy and even immunotherapy. This therapeutic pan-resistance, on top of other adaptive responses to targeted agents such as acquired mutations and lineage plasticity, underpins the current difficulties in eradicating cancer. In the present review, we attempt to provide a framework to understand the underlying biology of this major issue.

The pan-therapeutic resistance of disseminated tumor cells: Role of phenotypic plasticity and the metastatic microenvironment.

Cancer metastasis is the leading cause of mortality in patients with solid tumors. The majority of these deaths are associated with metastatic disease that occurs after a period of clinical remission, anywhere from months to decades following removal of the primary mass. This dormancy is prominent in cancers of the breast and prostate among others, leaving the survivors uncertain about their longer-term prognosis. The most daunting aspect of this dormancy and re-emergence is that the micrometastases in particular, and even large lethal outgrowths are often show resistance to agents to which they have not been exposed. This suggests that in addition to specific mutations that target single agents, there also exist adaptive mechanisms that provide this pan-resistance. Potential molecular underpinnings of which are the topic of this review.

Adult Stem Cell Functioning in the Tumor Micro-Environment.

Tumor progression from an expanded cell population in a primary location to disseminated lethal growths subverts attempts at cures. It has become evident that these steps are driven in a large part by cancer cell-extrinsic signaling from the tumor microenvironment (TME), one cellular component of which is becoming more appreciated for potential modulation of the cancer cells directly and the TME globally. That cell is a heterogenous population referred to as adult mesenchymal stem cells/multipotent stromal cells (MSCs). Herein, we review emerging evidence as to how these cells, both from distant sources, mainly the bone marrow, or local resident cells, can impact the progression of solid tumors. These nascent investigations raise more questions than they answer but paint a picture of an orchestrated web of signals and interactions that can be modulated to impact tumor progression.

Statins attenuate outgrowth of breast cancer metastases.

BACKGROUND: Metastasis in breast cancer foreshadows mortality, as clinically evident disease is aggressive and generally chemoresistant. Disseminated breast cancer cells often enter a period of dormancy for years to decades before they emerge as detectable cancers. Harboring of these dormant cells is not individually predictable, and available information suggests that these micrometastatic foci cannot be effectively targeted by existing therapies. As such, long-term, relatively non-toxic interventions that prevent metastatic outgrowth would be an advance in treatment. Epidemiological studies have found that statins reduce breast cancer specific mortality but not the incidence of primary cancer. However, the means by which statins reduce mortality without affecting primary tumor development remains unclear. METHODS: We examine statin efficacy against two breast cancer cell lines in models of breast cancer metastasis: a 2D in vitro co-culture model of breast cancer cell interaction with the liver, a 3D ex vivo microphysiological system model of breast cancer metastasis, and two independent mouse models of spontaneous breast cancer metastasis to the lung and liver, respectively. RESULTS: We demonstrate that statins can directly affect the proliferation of breast cancer cells, specifically at the metastatic site. In a 2D co-culture model of breast cancer cell interaction with the liver, we demonstrate that atorvastatin can directly suppress proliferation of mesenchymal but not epithelial breast cancer cells. Further, in an ex vivo 3D liver microphysiological system of breast cancer metastasis, we found that atorvastatin can block stimulated emergence of dormant breast cancer cells. In two independent models of spontaneous breast cancer metastasis to the liver and to the lung, we find that statins significantly reduce proliferation of the metastatic but not primary tumor cells. CONCLUSIONS: As statins can block metastatic tumor outgrowth, they should be considered for use as long-term adjuvant drugs to delay clinical emergence and decrease mortality in breast cancer patients.

A Model of Dormant-Emergent Metastatic Breast Cancer Progression Enabling Exploration of Biomarker Signatures.

Breast cancer mortality predominantly results from dormant micrometastases that emerge as fatal outgrowths years after initial diagnosis. In order to gain insights concerning factors associated with emergence of liver metastases, we recreated spontaneous dormancy in an all-human ex vivo hepatic microphysiological system (MPS). Seeding this MPS with small numbers (<0.05% by cell count) of the aggressive MDA-MB-231 breast cancer cell line, two populations formed: actively proliferating ("growing"; EdU+), and spontaneously quiescent ("dormant"; EdU-). Following treatment with a clinically standard chemotherapeutic, the proliferating cells were eliminated and only quiescent cells remained; this residual dormant population could then be induced to a proliferative state ("emergent"; EdU+) by physiologically-relevant inflammatory stimuli, lipopolysaccharide (LPS) and epidermal growth factor (EGF). Multiplexed proteomic analysis of the MPS effluent enabled elucidation of key factors and processes that correlated with the various tumor cell states, and candidate biomarkers for actively proliferating (either primary or secondary emergence) versus dormant metastatic cells in liver tissue. Dormancy was found to be associated with signaling reflective of cellular quiescence even more strongly than the original tumor-free liver tissue, whereas proliferative nodules presented inflammatory signatures. Given the minimal tumor burden, these markers likely represent changes in the tumor microenvironment rather than in the tumor cells. A computational decision tree algorithm applied to these signatures indicated the potential of this MPS for clinical discernment of each metastatic stage from blood protein analysis.

Liver 'organ on a chip'.

The liver plays critical roles in both homeostasis and pathology. It is the major site of drug metabolism in the body and, as such, a common target for drug-induced toxicity and is susceptible to a wide range of diseases. In contrast to other solid organs, the liver possesses the unique ability to regenerate. The physiological importance and plasticity of this organ make it a crucial system of study to better understand human physiology, disease, and response to exogenous compounds. These aspects have impelled many to develop liver tissue systems for study in isolation outside the body. Herein, we discuss these biologically engineered organoids and microphysiological systems. These aspects have impelled many to develop liver tissue systems for study in isolation outside the body. Herein, we discuss these biologically engineered organoids and microphysiological systems.

Inflammatory cytokine IL-8/CXCL8 promotes tumour escape from hepatocyte-induced dormancy.

BACKGROUND: Breast cancers can recur after a long latency period following 'successful' primary treatments. Chronic inflammation significantly correlates with reduced diseased-free survival in breast cancer patients and could be a point of intervention to prevent recurrence. Liver is among the main sites of breast cancer recurrence. Thus, we hypothesise that inflammatory signals from hepatic stellate cells, the major inflammatory regulators in the sinusoid, could stimulate dormant cancer cells to emerge. METHODS: We utilise in vitro co-culture of breast cancer cells with stellate cells and an ex vivo 3D human liver micro-physiologic system to identify stellate cells-derived factors that mediate tumour emergence. RESULTS: Activated, but not quiescent, hepatic stellate cells secreted soluble factors to induce the proliferation of MCF7 and MDA-MB231 cancer cells. IL-8 and MCP-1 were highly secreted by the activated stellate cells and primary human non-parenchymal cells. IL-8 significantly reduced serum-starvation growth arrest on MDA-MB231 cells in vitro and increased cancer proliferation ex vivo. Blocking IL-8Rb/CXCR2 reduced IL-8-induced cancer growth and proliferation. CONCLUSIONS: Activated stellate cells can induce breast cancer emergence from dormancy in the liver by secreting inflammatory cytokines. Preventing liver inflammation or disrupting the subsequent key cytokines may prevent metastatic outgrowth.

Bi-directional exosome-driven intercommunication between the hepatic niche and cancer cells.

BACKGROUND: Our understanding of the multiple roles exosomes play during tumor progression is still very poor and the contribution of the normal tissue derived exosomes in distant seeding and tumor outgrowth has also not been widely appreciated. METHODS: Using our all-human liver microphysiological system (MPS) platform as a model to closely recapitulate the early metastatic events, we isolated exosomes from both tumor cells and liver microenvironment. RESULTS: We observed that while priming of the hepatic niche (HepN) with MDA-231 breast cancer derived exosomes facilitated seeding of the cancer cells in the liver, subsequent tumor outgrowth was diminished; this was consistent with increased entry into dormancy. We found that hepatic niche (HepN) derived exosomes contribute significantly to the exosome pool and are distinguished from cancer derived exosomes based on their size, protein and miRNA content. By Ingenuity Pathway Analysis (IPA) of the miRNA content of the HepN, MDA-231/HepN and MDA-231 cells we showed that the HepN derived exosomes affect the breast cancer cells by suppressing pathways involved in cancer cell proliferation and invasion. More importantly exposure of MDA-231 and MDA-468 cells to purified normal HepN derived exosomes, induced changes in the cells consistent with a Mesenchymal to Epithelial reverting Transition (MErT). miRNA arrays performed on MDA-231 treated with Hum Hep/NPC derived exosomes showed significant changes in the levels of a select number of miRNAs involved in epithelial cell differentiation and miRNAs, such as miR186, miR23a and miR205, from our top and bottom bins have previously been reported to regulate E-cadherin transcription and MErT induction in various cancer types. Consistently HepN derived exosome treatment of breast and prostate cancer lines lead to a transient induction of E-cadherin and ZO-1 at the protein level and a more epithelial-like morphology of the cells. CONCLUSIONS: Collectively our data revealed a novel mechanism of regulation of the metastatic cascade, showing a well-orchestrated, timely controlled crosstalk between the cancer cells and the HepN and implicating for the first time the normal tissue/HepN derived exosomes in enabling seeding and entry into dormancy of the cancer cells at the metastatic site.

Liver protects metastatic prostate cancer from induced death by activating E-cadherin signaling.

Liver is one of the most common sites of cancer metastasis. Once disseminated, the prognosis is poor as these tumors often display generalized chemoresistance, particularly for carcinomas that derive not from the aerodigestive tract. When these cancers seed the liver, the aggressive cells usually undergo a mesenchymal to epithelial reverting transition that both aids colonization and renders the tumor cells chemoresistant. In vitro studies demonstrate that hepatocytes drive this phenotypic shift. However, the in vivo evidence and the molecular signals that protect these cells from induced death are yet to be defined. Herein, we report that membrane surface E-cadherin-expressing prostate cancer cells were resistant to cell death by chemotherapeutic drugs but E-cadherin null cells or those expressing E-cadherin only in the cytoplasm were sensitive to death signals and chemotherapies both in vitro and in vivo. While cell-cell E-cadherin ligandation reduced mitogenesis, this chemoprotection was proliferation-independent as killing of both 5-ethynyl-2'-deoxyuridine-positive (or Ki67+ ) and 5-ethynyl-2'-deoxyuridine-negative (Ki67- ) cells was inversely related to membrane-bound E-cadherin. Inhibiting the canonical survival kinases extracellular signal-regulated protein kinases, protein kinase B, and Janus kinase, which are activated by chemotherapeutics in epithelial cell-transitioned prostate cancer, abrogated the chemoresistance both in cell culture and in animal models of metastatic cancer. For disseminated tumors, protein kinase B disruption in itself had no effect on tumor survival but was synergistic with chemotherapy, leading to increased killing. CONCLUSION: Liver microenvironment-driven phenotypic switching of carcinoma cells and subsequent survival signaling results in activation of canonical survival pathways that protect the disseminated prostate cancer liver micrometastases in a proliferation-independent manner, and these pathways can be targeted as an adjuvant treatment to improve the efficacy of traditional chemotherapeutics (Hepatology 2016;64:1725-1742).

Macrophage phenotypic subtypes diametrically regulate epithelial-mesenchymal plasticity in breast cancer cells.

BACKGROUND: Metastatic progression of breast cancer involves phenotypic plasticity of the carcinoma cells moving between epithelial and mesenchymal behaviors. During metastatic seeding and dormancy, even highly aggressive carcinoma cells take on an E-cadherin-positive epithelial phenotype that is absent from the emergent, lethal metastatic outgrowths. These phenotypes are linked to the metastatic microenvironment, though the specific cells and induction signals are still to be deciphered. Recent evidence suggests that macrophages impact tumor progression, and may alter the balance between cancer cell EMT and MErT in the metastatic microenvironment. METHODS: Here we explore the role of M1/M2 macrophages in epithelial-mesenchymal plasticity of breast cancer cells by coculturing epithelial and mesenchymal cells lines with macrophages. RESULTS: We found that after polarizing the THP-1 human monocyte cell line, the M1 and M2-types were stable and maintained when co-cultured with breast cancer cells. Surprisingly, M2 macrophages may conferred a growth advantage to the epithelial MCF-7 cells, with these cells being driven to a partial mesenchymal phenotypic as indicated by spindle morphology. Notably, E-cadherin protein expression is significantly decreased in MCF-7 cells co-cultured with M2 macrophages. M0 and M1 macrophages had no effect on the MCF-7 epithelial phenotype. However, the M1 macrophages impacted the highly aggressive mesenchymal-like MDA-MB-231 breast cancer cells to take on a quiescent, epithelial phenotype with re-expression of E-cadherin. The M2 macrophages if anything exacerbated the mesenchymal phenotype of the MDA-MB-231 cells. CONCLUSION: Our findings demonstrate M2 macrophages might impart outgrowth and M1 macrophages may contribute to dormancy behaviors in metastatic breast cancer cells. Thus EMT and MErT are regulated by selected macrophage phenotype in the liver metastatic microenvironment. These results indicate macrophage could be a potential therapeutic target for limiting death due to malignant metastases in breast cancer.

Liver metastases: Microenvironments and ex-vivo models.

The liver is a highly metastasis-permissive organ, tumor seeding of which usually portends mortality. Its unique and diverse architectural and cellular composition enable the liver to undertake numerous specialized functions, however, this distinctive biology, notably its hemodynamic features and unique microenvironment, renders the liver intrinsically hospitable to disseminated tumor cells. The particular focus for this perspective is the bidirectional interactions between the disseminated tumor cells and the unique resident cell populations of the liver; notably, parenchymal hepatocytes and non-parenchymal liver sinusoidal endothelial, Kupffer, and hepatic stellate cells. Understanding the early steps in the metastatic seeding, including the decision to undergo dormancy versus outgrowth, has been difficult to study in 2D culture systems and animals due to numerous limitations. In response, tissue-engineered biomimetic systems have emerged. At the cutting-edge of these developments are ex vivo 'microphysiological systems' (MPS) which are cellular constructs designed to faithfully recapitulate the structure and function of a human organ or organ regions on a milli- to micro-scale level and can be made all human to maintain species-specific interactions. Hepatic MPSs are particularly attractive for studying metastases as in addition to the liver being a main site of metastatic seeding, it is also the principal site of drug metabolism and therapy-limiting toxicities. Thus, using these hepatic MPSs will enable not only an enhanced understanding of the fundamental aspects of metastasis but also allow for therapeutic agents to be fully studied for efficacy while also monitoring pharmacologic aspects and predicting toxicities. The review discusses some of the hepatic MPS models currently available and although only one MPS has been validated to relevantly modeling metastasis, it is anticipated that the adaptation of the other hepatic models to include tumors will not be long in coming.

Immunomodulatory activity of Melaleuca alternifolia concentrate (MAC): inhibition of LPS-induced NF-κB activation and cytokine production in myeloid cell lines.

Melaleuca alternifolia concentrate (MAC) is a mixture predominantly composed of monoterpenoids and sesquiterpenes, refined from the essential oil of the tea tree by removing up to 99% of the more toxic, hydrophobic monoterpenes. MAC was examined here for its immunomodulatory effects on the human THP1 and murine RAW264.7 myeloid leukemic cell lines as models for macrophage-like cells. Firstly, MAC levels were determined that did not affect either the survival or proliferation of these cell lines in vitro. Next, the levels of lipopolysaccharide (LPS)-induced production of cytokines (IL-6, TNFα, IL-10, GM-CSF, IFNγ and IL-3) were examined from the myeloid cell lines using multiplex assays. Many of the LPS-inducible cytokines produced by either cell lines could be significantly inhibited by MAC. Closer examination of the mechanism of action of MAC showed that it inhibited the LPS-induced activation of IκB phosphorylation and nuclear factor (NF)-κB signalling and translocation, inhibiting iNOS protein expression and NO production. These results demonstrate that MAC exerts its immunomodulatory effects by inhibiting NF-κB signalling activation and levels of cytokine production by macrophage-like cell lines.